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Method S1.
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Characterization of monocultures of protozoa by 18S rRNA sequencing
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Protozoa were grown in 100ml-portions of cereal grass medium, concentrated by
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centrifugation at 300 x g for 10 min and the cell pellet was used to extract template DNA
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used for the amplification of 18S rRNA sequences. Template DNA was obtained by
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disrupting the protozoan cells mixed with an equal amount of 0.1-mm silica-zirconium
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beads in a mini-bead beater (BioSpec Products, Bartlesville, OK). The tubes with
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protozoa-bead mixture were shaken five times of 60 sec pulses alternating with 60 sec
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cooling on ice. The cell debris was pelleted by centrifugation and the supernatant
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containing the DNA was used to amplify 18S rRNA as described by Karnati et al. [1]
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using the protozoa-specific forward primer P-SSU-342 (5’-
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CTTTCGATGGTAGTGTATTGGACTAC-3’) and a eukarya-specific reverse primer
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Medlin B (5’-TGATCCTTCTGCAGGTTCACCTAC-3’) with the following
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modification. Briefly, genomic DNA was amplified in ten 50-µl reaction mixtures
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containing 5 to 100 ng of template DNA, 500 nM of each primer and two Illustra
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PuReTaq Ready-To-Go PCR Beads (GE Healthcare, Piscataway, NJ). PCR reactions
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were carried out using DNA Engine Dyad® Peltier Thermal Cycler (Bio-Rad labs.,
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Hercules, CA) using the following parameters: one cycle of 94OC for 2 min; 30 cycles of
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94OC for 60 sec, 37OC for 65 sec, and 72OC for 3 min; and a final 6-min extension at
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72OC. PCR products were purified with DNA Clean and Concentrator-5 kit (Zymo
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Research, Orange, CA) by using 5 volumes of DNA binding buffer to each volume of
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PCR product mixture as per manufacture’s instructions. PCR products were cloned using
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the TOPO TA cloning kit as per the manufacturer’s instructions and transformed into E.
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coli TOP10F’ One Shot competent cells (Invitrogen). Two PCR reactions were
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performed for each protozoan, and 5 clones were picked from each PCR to minimize
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potential PCR bias. DNA templates for sequencing were prepared using the Illustra
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Templiphi 100 amplification kit (GE Healthcare) as described by the manufacturer.
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Sequencing reactions were performed as per the manufacturer’s instructions for the Big
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Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using
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the Medlin B [1] reverse primer. Cycle sequencing reactions were purified using DyeEx
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96 kit (Qiagen, Valencia, CA) and sequencing was carried out using an Applied
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Biosystems 3730 DNA analyzer. DNA sequences were trimmed at both the 5’ and 3’
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ends to correct falsely called bases by using Lasergene SeqMan Pro (v7.0, DNASTAR,
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Inc., Madison, WI). Only sequences with unambiguous reads of >500 bp were used; each
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read used averaged approximately 600 bp. The compiled sequence data sets were aligned
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with the closest sequence relatives from the GeneBank database by using Kodon (v3.5,
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Applied Maths, Inc., Austin, TX).
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REFERENCE
1. Karnati SK, Yu Z, Sylvester JT, Dehority BA, Morrison M, et al. (2003) Technical
note: Specific PCR amplification of protozoal 18S rDNA sequences from DNA
extracted from ruminal samples of cows. J Anim Sci 81: 812-815.